1. Volume 16, Issue 7, Pages 1874-1890 (August 2016)
Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly 
Oliver Rackham, Jakob D. Busch, Stanka Matic, Stefan J. Siira, Irina Kuznetsova, Ilian Atanassov, Judith A. Ermer, Anne-Marie J. Shearwood, Tara R. Richman, James B. Stewart, Arnaud Mourier, Dusanka Milenkovic, Nils-Göran Larsson, Aleksandra Filipovska 
Cell Reports 
Volume 16, Issue 7, Pages (August 2016)
DOI: /j.celrep
Copyright © 2016 The Author(s) Terms and Conditions

2. Cell Reports 2016 16, 1874-1890DOI: (10.1016/j.celrep.2016.07.031)
Copyright © 2016 The Author(s) Terms and Conditions

3. Figure 1
Full-Body Knockout of MRPP3 Causes Embryonic Lethality and Cardiomyopathy in Heart-Specific Knockout Mice
(A) Schematic representation of the gene organization in mammalian mtDNA.
(B) Schematic showing the disruption of the Mrpp3 gene (also known as LRik and Kiaa0391). LoxP sites flanking exon 3 of the Mrpp3 gene were inserted in the mouse genome by homologous recombination. The long homology arm (LHA), floxed regions (FR), and short homology arm (SHA) are shown.
(C) Homologous recombination at the Mrpp3 locus shown by Southern blotting.
(D) Morphology of the Mrpp3+/+ and Mrpp3−/− embryos at day E8.5. Scale bar, 1 mm.
(E) Heart weight to body weight ratio in control (L/L) and knockout mice (L/L, cre) at different ages. At 4 weeks, L/L n = 8, L/L, cre n = 8; at 8 weeks, L/L n = 12, L/L, cre n = 12; at 11 weeks, L/L n = 12, L/L, cre n = 12. Error bars, SEM; ∗∗p < 0.01; ∗∗∗p < 0.001, Student’s t test.
(F) H&E, NADH, and COX and trichrome gomorii staining of hearts from control (L/L) and knockout mice (L/L, cre).
(G) Levels of Atf4, Atf5, and Chop mRNAs were determined in isolated total heart RNA from control (L/L) and knockout mice (L/L, cre) by qRT-PCR (n = 5). The data are expressed relative to control mice and normalized to 18S rRNA.
(H) Mrpp3 transcript levels were determined in isolated total heart RNA from control (L/L) and knockout mice (L/L, cre) by qRT-PCR (n = 5). The data are expressed relative to control mice and normalized to 18S rRNA.
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4. Figure 2
Loss of MRPP3 Impairs Mitochondrial Biogenesis and Increases Transcription
(A) In organello transcription was measured in heart mitochondria in the presence of 32P-labeled UTP, and the radiolabeled RNA was isolated and resolved on a 0.9% formaldehyde gel that was visualized by autoradiography. VDAC was used as a loading control. The data are representative of results obtained for 11-week-old L/L and L/L, cre mice (n = 6) in three independent biological experiments.
(B) Protein synthesis in heart mitochondria from 11-week-old control and knockout mice was measured by pulse incorporation of 35S-labeled methionine and cysteine. Equal amounts of mitochondrial protein (50 μg) were separated by SDS-PAGE, immunoblotted for VDAC to show equal loading and visualized by autoradiography. Representative gels are shown of three independent biological experiments.
(C) Mitochondrial proteins (25 μg) from 4-, 8-, and 11-week-old heart mitochondria from control and knockout mice were resolved on 4%–20% SDS-PAGE gels and immunoblotted against antibodies to investigate the steady-state levels of nuclear and mitochondrial-encoded proteins. ATP5A was used as a loading control.
(D) Heart mitochondria (75 μg) treated with 1% digitonin were separated on a 4%–30% BN-PAGE, and the respiratory complexes were visualized by Coomassie staining. In-gel activity stains were used for complex I and complex IV.
(E) Respiratory complex activities were measured spectrophotometrically and normalized to citrate synthase activity in mitochondria isolated from hearts of 11-week-old control and knockout mice (n = 6). Error bars, SEM; ∗∗p < 0.01; ∗∗∗p < 0.001, Student’s t test.
(F) Loss of the MRPP3 causes profound reduction in mitochondrial respiration at complex I, II-III, and IV in the KO mice compared to controls. Non-phosphorylating (state 4), phosphorylating (state 3), and uncoupled respiration in the presence of 0.5 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was measured in heart mitochondria using an Oroboros oxygen electrode using pyruvate, glutamate and malate, or succinate as a substrates in the presence of rotenone as indicated. Error bars, SEM; ∗∗p < 0.01; ∗∗∗p < 0.001, Student’s t test.
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5. Figure 3 Loss of MRPP3 Causes Impaired RNA Processing
(A–C) The abundance of mature mitochondrial mRNAs (A), tRNAs (B), and rRNAs (C) in hearts from 4-, 8-, and 11-week-old control and knockout mice were analyzed by northern blotting. 18S rRNA was used as a loading control. The data are representative of results obtained from at least eight mice from each strain and three independent biological experiments.
(D) The canonical mitochondrial RNA junctions were measured in total heart RNA from control (L/L) and knockout (L/L, cre) mice by qRT-PCR and normalized to 18S rRNA. Error bars, SEM; ∗∗p < 0.01; ∗∗∗p < 0.001, Student’s t test.
(E) Frequency distribution of PARE reads from heart mitochondrial RNA from three control (L/L; blue) and three knockout (L/L, cre; red) mice. Windows are centered on reads that align 15 nt away from either side of all annotated 5′ ends of mitochondrial tRNAs from the heavy and light chain.
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6. Figure 4
Transcriptome-wide Analyses of 5′ tRNA Cleavage Sites by PARE and RNA-Seq
(A) A complete map of changes in 5′ end abundance (1, 5; log2 fold change[KOmean/Ctrlmean]) and RNA-seq coverage (2, 4; log2 fold change[KOmean/Ctrlmean]) from three control (L/L) and three knockout (L/L, cre) mice, on heavy (1, 2) and light (4, 5) strands. Increases are shown in red and decreases in blue. The mitochondrial genome is displayed in the central track (3), with the nucleotide position in base pairs displayed across the exterior; rRNAs are displayed in orange, mRNAs in green, tRNAs in blue, and the non-coding region (NCR) in gray.
(B) Genome browser view of the 5′ end abundance from three control (L/L) and three knockout (L/L, cre) mice (top; mean normalized count), and relative changes in mean 5′ end abundance (middle; log2 fold change[KOmean/Ctrlmean]), and mean RNA-seq coverage (bottom; log2 fold change[KOmean/Ctrlmean]) showing the 5′ cleavage sites of mt-tRNAVal by MRPP3 and the downstream effect on the 3′ end of the 12S rRNA in the absence of MRPP3. Regions of interests are shown in gray (for 5′ ends) and green (for 3′ ends) boxes.
(C) Genome browser view of the 5′ end abundance from three control (L/L) and three knockout (L/L, cre) mice (top; mean normalized count), and relative changes in mean 5′ end abundance (middle; log2[KOmean/Ctrlmean]), and mean RNA-seq coverage (bottom; log2[KOmean/Ctrlmean]) showing the 5′ cleavage sites of mt-tRNALeu(UUR) by MRPP3 and the downstream effect on the 3′ end of the mt-Nd1 mRNA in the absence of MRPP3. Regions of interests are shown in gray (for 5′ ends) and green (for 3′ ends) boxes.
(D) Genome browser view of the 5′ end abundance from three control (L/L) and three knockout (L/L, cre) mice (top; mean normalized count), and relative changes in mean 5′ end abundance (middle; log2 fold change[KOmean/Ctrlmean]), and mean RNA-seq coverage (bottom; log2 fold change[KOmean/Ctrlmean]) showing the non-canonical region between mt-Atp8/6 and mt-Co3 and the 5′ cleavage sites of mt-tRNALys by MRPP3 and the lack of effect on the 3′ end of the mt-Atp8/6 mRNA in the absence of MRPP3. Regions of interests are shown in gray (for 5′ ends) and green (for 3′ ends) boxes.
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7. Figure 5
Impaired RNA Processing Causes an Imbalance in Nuclear-Encoded Mitochondrial Proteins
Transcriptome-wide RNA-seq was carried out on total heart RNA from three control (L/L) and three knockout (L/L, cre) mice, and differential expression analyses were performed.
(A and B) Significant differences (p < 0.05) in positively and negatively correlated transcripts relative to controls are based on specific gene ontologies are shown for cardiac markers and mitochondrial genes (A) and for oxidative phosphorylation, the translation machinery, and mitoribosomal and RNA-binding proteins (B).
(C) The levels of nuclear-encoded mitochondrial RNA-binding proteins were measured by immunoblotting in heart mitochondria from 4-, 8-, and 11-week-old control (L/L) and knockout (L/L, cre) mice.
(D) Mitochondrial ribosomal protein abundance was measured by immunoblotting in heart mitochondria from 4-, 8-, and 11-week-old L/L and L/L, cre mice.
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8. Figure 6
Mitochondrial Ribosome Assembly Is Impaired as a Consequence of Impaired RNA Processing
(A) A continuous 10%–30% sucrose gradient was used to determine the distribution of the small and large ribosomal subunit and the monosome in the control and knockout mice. Mitochondrial ribosomal protein markers of the small (MRPS35) and large (MRPL37, MRPL44, MRPL11, and MRPL12) ribosomal subunits were detected by immunoblotting with specific antibodies. The input (10% of the total lysate) was used as a positive control. The data are representative of results from at least three independent biological experiments.
(B and C) The distribution of the mt-Co1 mRNA, 12S and 16S rRNAs (B), and 12S-16S rRNA junctions (C) in sucrose gradients were analyzed by qRT-PCR. The data are expressed as percentage of total RNA abundance and are representative of results from four independent biological experiments.
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9. Figure 7
Differential Distribution of Mitochondrial Ribosomal Proteins upon Loss of MRPP3
Mass spectrometry was used to analyze the distribution of the mitochondrial ribosomal proteins in sucrose gradient fractions that contained the small (fraction 8) and large ribosomal subunit (fraction 10) and the monosome (fractions 12 and 13) in the control (L/L) mice and compared to the same fractions in the Mrpp3 knockout mice (L/L, cre), both at 11 weeks. The analyses were performed on fractions from three separate biological experiments from three control and three knockout mice (n = 3). The results of the ANOVA analysis were adjusted for multiple testing using the Benjamini and Hochberg procedure. Only proteins with adjusted p value of less than 0.01 (1% FDR) were termed significant and marked with asterisk (∗).
Cell Reports  , DOI: ( /j.celrep )
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